The human major histocompatibility complex (MHC) contains diverse genes functioning in the processing and presentation of antigen-derived peptides to T-cells. Class I genes encode molecules that bind peptides in the endoplasmic reticulum (ER) and display these on the cell surface to cytotoxic T-cells. This function is dependent on other MHC genes involved in the generation of the peptides in the cytosol and their transport into the lumen of the ER. Recent functional and genetic evidence indicates that this pathway is dependent on an additional yet unidentified gene(s) linked to the MHC. Moreover, novel MHC class I genes probably serving a specialized function in antigen presentation have been discovered. The following studies are proposed: Specific Aim I: Class I molecules physically interact with the MHC-encoded transporter associated with antigen processing (TAP), which delivers the peptides that are mainly bound by class I molecules from the cytosol into the ER. A novel defect in a human mutant cell line completely abrogates this interaction and thus greatly impairs the ability of class I molecules to bind peptides. This deficiency results from loss of function of an unidentified gene(s) linked to the MHC. The identification of this gene(s) is the main goal of the proposed research. This will be accomplished using molecular techniques for cDNA and genomic DNA subtraction and cloning. Isolated candidate gene sequences will be screened for their specific association with the mutant phenotype, and transfection of cosmids and of cDNA constructs will be used to restore normal function in the mutant cell line. The identified gene(s) will be sequenced, and the function and cell biology of the encoded protein will be investigated. Moreover, T-cell assays and molecular biology experiments are proposed to further characterize the functional defect in the mutant cell line. Specific Aim II: Novel MHC class I genes, MICA and MICB encoding highly diverged yet evolutionary conserved class I heavy chains have been identified. These genes are specifically transcribed in epithelial cells and are regulated by the cell-stress response system. The MICA chain is exclusively expressed at high levels on the surface of epithelial cells. The proposed studies will define the antigen processing and assembly pathways employed by this unusual MHC class I molecule.